Methods for treating or preventing ischemic injury

ABSTRACT

A therapeutic or prophylactic treatment method of myocardial ischemia, such as due to myocardial infarction, by administering erythropoietin, alone or in combination with other drugs, to a patient suffering from or at risk of cardiac injury, such as myocardial ischemia. The erythropoietin is administered in a concentration such that the subject&#39;s hematocrit level or production of red blood cells is not significantly affected.

FIELD OF THE PRESENT INVENTION

The present invention generally relates to methods, preparations andpharmaceutical compositions for treating or preventing ischemic injuryin mammalian subjects. More specifically, the present invention useserythropoietins to treat myocardial ischemia or ischemia-reperfusioninjury in patients in need thereof.

DESCRIPTION OF THE RELATED ART

Ischemia occurs when the flow of blood to a region of the body isdecreased or eliminated, such as during a myocardial infarction, causingdamage to the tissue distal to the blockage. In the United States,approximately 1.5 million myocardial infarctions (MIs) occur each year,and mortality with acute infarction is approximately 30 percent(Pasternak, R. and Braunwald, E., Acute Myocardial Infarction,HARRISON'S PRINCIPLES OF INTERNAL MEDICINE, 13th Ed., McGraw Hill Inc.,p.p. 1066-77 (1994)). Myocardial infarction occurs generally with anabrupt decrease in coronary blood flow that follows a thromboticocclusion of a coronary artery. The occluded artery often has beennarrowed previously by atherosclerosis, and the risk of recurrentnonfatal myocardial infarction persists in many patients. Ultimately,the extent of myocardial damage caused by the coronary occlusion dependsupon the “territory” supplied by the affected vessel, the degree ofocclusion of the vessel, the amount of blood supplied by collateralvessels to the affected tissue, and the demand for oxygen of themyocardium whose blood supply has suddenly been limited (Pasternak, R.and Braunwald, E. Acute Myocardial Infarction, HARRISON'S PRINCIPLES OFINTERNAL MEDICINE, 13th Ed., McGraw Hill Inc., p.p. 1066-77 (1994)).

In some cases, the flow of blood to a region of the body is temporarilyhalted and then re-established (reperfusion), resulting inischemia-reperfusion injury. Ischemia-reperfusion injury can occurduring certain surgical procedures, such as repair of aortic aneurysmsand organ transplantation. Clinically, ischemia-reperfusion injury ismanifested by such complications as pulnonary dysfunction, includingadult respiratory distress syndrome, renal dysfunction, consumptivecoagulopathies including thrombocytopenia, fibrin deposition into themicrovasculature and disseminated intravascular coagulopathy, transientand permanent spinal cord injury, cardiac arrhythmias and acute ischemicevents, hepatic dysfunction including acute hepatocellular damage andnecrosis, gastrointestinal dysfunction including hemorrhage and/orinfarction and multisystem organ dysfunction (MSOD) or acute systemicinflammatory distress syndromes (SIRS). The injury may occur in theparts of the body to which the blood supply was interrupted, or it canoccur in parts fully supplied with blood during the period of ischemia.

Erythropoietin (also known as Epo, epoetin or procrit) is an acidicglycoprotein hormone of approximately 34,000 dalton molecular weightoccurring in multiple forms, including alpha, beta, omega and asialo.Erythropoietin stimulates red blood cell production. It is produced inthe kidney and stimulates the division and differentiation of committederythroid precursors in the bone marrow and elsewhere. Generally,erythropoietin is present in very low concentrations in plasma when thebody is in a healthy state, in which tissues receive sufficientoxygenation from the existing number of erythrocytes. This normal lowconcentration is enough to stimulate replacement of red blood cells thatare lost normally through aging. The amount of erythropoietin in thecirculation is increased under conditions such as hypoxia, when oxygentransport by blood cells in the circulation is reduced. Hypoxia may becaused by loss of large amounts of blood through hemorrhage, destructionof red blood cells by over-exposure to radiation, reduction in oxygenintake due to high altitudes or prolonged unconsciousness, or variousforms of anemia or ischemia. In response to tissues undergoing hypoxicstress, erythropoietin will increase red blood cell production bystimulating the conversion of primitive precursor cells in the bonemarrow into proerythroblasts which subsequently mature, synthesizehemoglobin and are released into the circulation as red blood cells.When the number of red blood cells in circulation is greater than neededfor normal tissue oxygen requirements, erythropoietin in circulation isdecreased.

Clinically, erythropoietin is used as a treatment for anemia associatedwith renal disease, cancer chemotherapy, malignancies, adult andjuvenile rheumatoid arthritis, disorders of hemoglobin synthesis,prematurity, and treatment of HIV infection. Erythropoietin is primarilyused to induce production of red blood cells to combat anemia. (See,e.g., Bottomley et al. (2002) Lancet Oncol. 3:145). Erythropoietin hasalso been suggested to be useful in controlling bleeding in patientswith abnormal hemostasis. (See e.g., U.S. Pat. No. 6,274,158).Recombinant human erythropoietin (rHuEpo or epoetin α) is commerciallyavailable as EPOGEN® (epoetin alfa, recombinant human erythropoietin)(Amgen Inc., Thousand Oaks, Calif.) and as PROCRIT® (epoetin alfa,recombinant human erythropoietin) (Ortho Biotech Inc., Raritan, N.J.).

The normal ranges for hematocrit values are 37-48 percent for women and42-52 percent for men. (See Case Records of the Massachusetts GeneralHospital: normal reference laboratory values. (1992) N. Eng. J. Med.327:718). However, hematocrit values in patients with renal failure aregenerally below 33 percent (See Besarab et al. (1998) N. Eng. J. Med.339: 584). Several studies have indicated that increasing hematocritlevels in hemodialysis patients from below 30 percent to 30 to 38percent increases cardiac function and other parameters, and that themortality rate in patients with hematocrits below 30 is higher thanpatients with hematocrits of 30 to 35 percent. Other studies have shownthat the use of erythropoietin to increase hematocrit levels in patientswith cardiovascular disease (congestive heart failure, coronary arterydisease or prior myocardial infarction) to within the normal range (42percent) results in a slight increase in adverse cardiac events (i.e.,death or a first non-fatal myocardial infarction) as compared tosubjects in which erythropoietin was used to increase hematocrit levelsto 30 percent. (See Besarab et al. (1998) N. Eng. J. Med. 339: 584). Intransgenic mice made polyglobulic (hematocrit of 80 percent) by theexogenous expression of erythropoietin, mortality due to cardiovascularcomplications was prevented by activation of the nitric oxide pathway.(See Ruschitzka et al. (2000) P.N.A.S. 97:11609). Thus the safety andefficacy of use of erythropoietin to increase hematocrit levels inpatients with cardiovascular disease, especially those suffering fromrenal failure, must be further evaluated. Also unexplored are theprotective and therapeutic effects of erythropoietin on thecardiovascular system independent of its hematopoietic activities.

SUMMARY OF THE INVENTION

The present invention relates to the discovery that myocardial oxidativeand/or nitrosative stress can be prevented or minimized byadministration of cardioprotective factors, and thus has benefit fortreating cardiovascular and other diseases. In particular, it has beenfound that erythropoietin is useful as a cardioprotective andcardiotherapeutic agent, and is therefore valuable in the treatment of avariety of various heart-related ailments such as myocardial infarction,ischemia-reperfusion injury, congestive heart failure, and cardiacarrest, and for cardioprotection. Erythropoietin in particular has beenfound to protect myocardial contractility in a subject suffering from acardiac injury without increasing hematocrit levels. Erythropoietin hasalso been found to preserve cardiac beta adrenergic receptor densityduring cardiac injury. These discoveries provide the means to improvepharmacologically the contractility of failing or otherwise impairedhearts, and provide both endogenous and pharmacological means to improvethe function of an impairment of heart rhythm or pump function.

The compositions and methods of the invention are surprisingly usefulfor the reduction or elimination of hypoxic/ischemic cardiac injury invivo and ex vivo, as well as the prevention and/or treatment ofcardiovascular disease in mammals in need thereof, such as humans.

In one aspect, the present invention relates to a method of treating orpreventing myocardial oxidative and/or nitrosative stress in a subjectby administering erythropoietin to a subject in need thereof at aconcentration that does not increase the hematocrit in said subject,such that one or more myocardial cells which are the subject of saidoxidative stress are protected from cell death. Myocardial oxidativestress may be caused by hypoxia, ischemia or other causes. Myocardialnitrosative stress may be caused by drugs, infection, inflammation,hypoxia, ischemia or other causes. Erythropoietin is typicallyadministered at a concentration or for a duration that will not inducered blood cell formation or alternatively, increase the hematocrit in asubject, e.g., between about 1 pM and less than 1000 μM, including lessthan 900 μM, less than 700 μM, less than 500 μM, less than 300 μM, lessthan 100 μM, or less than 50 μM. In other embodiments, erythropoietin isadministered as a function of the subject's body weight. Erythropoietinmay typically be administered at a concentration of between about 1 U/kgto 10,000 U/kg of a subject's body weight, including less than 7,500U/kg, 5,000 U/kg, 2500 U/kg, 1000 U/kg, 750 U/kg, 500 U/kg, 250 Ug/kg,100 Ug/kg, 50 U/kg, 25 U/kg, 10 U/kg, 5 U/kg, or 1 U/kg.

In another aspect, the present invention relates to a method of treatingor preventing myocardial oxidative and/or nitrosative stress in asubject by administering erythropoietin to a subject in need thereof ata concentration that does not induce red blood cell production in saidsubject, such that one or more myocardial cells which are the subject ofsaid oxidative or nitrosative stress are protected from cell death.

In another aspect, the present invention relates to a method ofmodulating a cardioprotective signaling pathway by administering, to asubject in need of cardioprotection, erythropoietin at a concentrationthat does not induce red blood cell production in the subject. Thisconcentration of erythropoietin is an amount effective to enhance ormaintain the effect of the cardioprotective signaling pathway. Thecardioprotective signaling pathways include MAP kinase, PI3 kinase, aninsulin-responsive pathway, hormones, ischemia preconditioning,adenosine pathways, ras, JAK/STAT, nitric oxide synthase, hemoxygenase,xanthine oxidase, NADPH oxidase, cytochrome p450, cytochrome p450reductase, oxigenases, denitrosylases, GSNO reductase, oxygen-carryingproteins, nitric oxide-carrying proteins, and carbon monoxide-carryingproteins.

In another aspect, the present invention relates to a method of treatingor preventing cardiac injury caused by hypoxia or ischemia in a subjectby administering erythropoietin to a subject in need thereof at aconcentration that does not increase the hematocrit in the subject, suchthat the hypoxia or ischemic-related injury is prevented or decreased.Cardiac injury can include myocardial infarction; cardiac arrest;ischemia-reperfusion injury; congestive heart failure; cardiotoxicity;cardiac damage due to parasitic infection; fulminant cardiacamyloidosis; heart surgery; heart transplantation; and traumatic cardiacinjury. The erythropoietin may be administered prior to, at the onsetof, or following cardiac injury.

In another aspect, the present invention relates to a method of treatingor preventing cardiac injury in a subject by administeringerythropoietin to a subject in need thereof at a concentration that doesnot induce red blood cell production in the subject.

In another aspect, the present invention relates to a method of treatingor preventing cardiac injury in a subject by administeringerythropoietin to a subject in need thereof for a time period in whichthe hematocrit of the subject is not increased.

In another aspect, the present invention relates to a method ofpreventing organ damage during organ or tissue transplantation byadministering to an organ donor erythropoietin at a concentration thatdoes not increase the hematocrit in the donor prior to or concurrentwith removal of the organ, such that damage caused by reperfusion of theorgan or tissue is decreased or prevented. Any organ or tissue capableof being transferred by medical procedures known in the art isencompassed by the present invention.

In another aspect, the present invention relates to a method of treatingheart failure in a subject by treating the subject with erythropoietinat a concentration that does not increase the hematocrit in the subjectand a compound selected from the group consisting of anti-plateletdrugs, anti-coagulant drugs, anti-thrombotic drugs.

In another aspect, the present invention relates to a method of treatinga survivor of a myocardial infarction by administering erythropoietin ata concentration that does not increase the hematocrit in a survivor,wherein the erythropoietin is administered in a single dose within 1hour of the myocardial infarction.

In another aspect, the present invention relates to a method of treatinga survivor of a myocardial infarction by administering erythropoietin ata concentration that does not increase the hematocrit in the survivor,wherein the erythropoietin is administered for an extended period oftime.

In another aspect, the present invention relates to a method ofpreventing or reducing the severity of ischemia-reperfusion injury in asubject at risk for ischemia-reperfusion injury by administering to thesubject an amount of erythropoietin at a concentration that does notincrease the hematocrit of the subject. In some embodiments of thepresent invention, the ischemia-reperfusion injury can be caused bysurgical repair of a thoracic aortic aneurysm, a suprarenal aorticaneurysm, liver, kidney, small intestine, or pancreas transplant,hepatic and biliary surgical resections, total or partialpancreatectomy, total and partial gastrectomy, esophagectomy, colorectalsurgery, vascular surgery for mesenteric vascular disease, abdominalinsufflation during laparoscopic surgical procedures, blunt orpenetrating trauma to the abdomen including gun shot wounds, stab woundsor penetrating wounds or blunt abdominal trauma secondary todeceleration injury or motor vehicle accidents, hemorrhagic shock due toblood loss, cardiogenic shock due to myocardial infarction or cardiacfailure, neurogenic shock or anaphylaxis.

In another aspect, the present invention relates to a method ofpreconditioning a subject at risk for a cardiac injury due to a surgicalprocedure by administering erythropoietin at a concentration that doesnot increase the hematocrit in the subject.

In another aspect, the present invention relates to a method ofpreconditioning a subject at risk for a cardiac injury due to a surgicalprocedure by administering erythropoietin for a time period in which thehematocrit in the subject is not increased.

In another aspect, the present invention relates to a method ofincreasing beta-receptor density in a subject suffering from cardiacinjury by administering erythropoietin at a concentration that does notincrease the hematocrit in the subject.

In another aspect, the present invention relates to a method ofpreserving or increasing beta-receptor sensitivity in a subjectsuffering from cardiac injury by administering erythropoietin at aconcentration that does not increase the hematocrit in the survivor,such that beta-receptor sensitivity is not substantially decreased.

In another aspect, the present invention relates to a method ofpreventing reduced sensitivity to one or more cardiostimulatorycompounds in a subject suffering from or at risk of a cardiac injury byadministering erythropoietin at a concentration that does not increasethe hematocrit in the subject such that the patients' reaction to saidone or more cardiostimulatory compounds is not reduced over time.

In another aspect, the present invention relates to a method ofincreasing beta-receptor density in a subject suffering from cardiacinjury by administering erythropoietin for a time period in which thehematocrit in the subject is not increased.

These and other objects of the present invention will be apparent fromthe detailed description of the invention provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the protection of cardiac contractility whenerythropoietin (EPO) is administered 5 minutes after suture ligation ofthe large branch of the left circumflex artery (LCx). MI only, LCxligation alone; EPO, 5000 U/kg of EPO administered following LCxligation. Basal contractility, at rest; Iso-Low, Isoproterenol 0.033μg/kg/min; Iso-High, Isoproterenol 0.1 μg/kg/min.

FIG. 2 depicts the protection of cardiac contractility whenerythropoietin (EPO) is administered 24 hous prior to suture ligation ofthe large branch of the left circumflex artery (LCx). MI only, LCxligation alone; EPO, 5000 U/kg of EPO administered 24 hous prior to LCxligation. Basal contractility, at rest; Iso-Low, Isoproterenol 0.033μg/kg/min; Iso-High, Isoproterenol 0.1 μg/kg/min.

FIG. 3 depicts a comparison of the hematocrit data from animalsreceiving systemic normal saline (Control) versus Erythropoietin (EPO5,000 U/kg).

FIG. 4 depicts a comparison of left ventricular B-receptor density in 3groups: LCx ligation alone (MI Only); Administration of erythropoietin(5000 U/kg) followed by LCx ligation 24 hous later (EPO); and untreatedgroup (Sham).

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention will now be moreparticularly described with reference to the accompanying drawings andpointed out in the claims. It will be understood that particularembodiments described herein are shown by way of illustration and not aslimitations of the invention. The principal features of this inventioncan be employed in various embodiments without departing from the scopeof the invention. All parts and percentages are by weight unlessotherwise specified.

Definitions

For convenience, certain terms used in the specification, examples, andappended claims are collected here. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention pertains. However, to the extent that these definitions varyfrom meanings circulating within the art, the definitions below are tocontrol.

“Erythropoietin” includes any variants, fragments, conjugates,derivatives, and mutants of the erythropoietin protein, produced bynatural, recombinant or synthetic means.

“Ischemia” includes the decrease or cessation of myocardial blood flow.

“Hypoxia” includes the deficiency in the amount of oxygen reaching bodytissues.

“Hypoxia or ischemic-related injury” includes cardiac injury.

“Reperfusion” includes the restoration of blood flow to an organ ortissue that has had its blood supply cut off, as after a heart attack orstroke.

“Oxidative stress” includes conditions that occur when there is anexcess of free radicals, a decrease in antioxidant levels, or both.

“Nitrosative stress” includes impetus for NO or NO₂ group attachment toproteins, nucleic acids or other biological molecules. Nitrosativestress is distinct from oxidative stress and can occur under anaerobicconditions. Nitrosative stress can be caused by an increase innitrosation or nitrosating agents, or a decrease in anti-nitrosants, ora combination of these factors.

“Cardiac stunning” includes cardiac contractile dysfunction, such as dueto surgical procedures, and may include troponin-I dysfunction.

“Necrosis” includes the death of cells or tissues through injury ordisease, particularly in a localized area of the body such as themyocardium.

“Apoptosis” refers to programmed cell death.

“Sensitivity” refers in part to the efficacy of a treatment.

“Anti-arrhythmic compounds” include any compounds useful in treating anirregular or abnormal heartbeat.

“Contractility enhancing compounds” include any compounds that increasemyocardial contraction.

“Beta blockers” include agents such as atenolol, metoprolol, andpropranolol, which act as competitive antagonists at the adrenergic betareceptors. Such agents also include those more selective for the cardiac(beta-1) receptors which allows for decreased systemic side effects.Beta blockers reduce the symptoms connected with hypertension, cardiacarrhythmias, migraine headaches, and other disorders related to thesympathetic nervous system. Beta blockers also are sometimes given afterheart attacks to stabilize the heartbeat. Within the sympathetic nervoussystem, beta-adrenergic receptors are located mainly in the heart,lungs, kidneys, and blood vessels. Beta blockers compete with thenerve-stimulating hormone epinephrine for these receptor sites and thusinterfere with the action of epinephrine, lowering blood pressure andheart rate, stopping arrhythmias, and preventing migraine headaches.

“Cardiac injury” includes any chronic or acute pathological eventinvolving the heart and/or associated tissue (e.g., the pericardium,aorta and other associated blood vessels), includingischemia-reperfusion injury; congestive heart failure; cardiac arrest;myocardial infarction; cardiotoxicity caused by compounds such as drugs(e.g., doxorubicin, herceptin, thioridazine and cisapride); cardiacdamage due to parasitic infection (bacteria, fungi, rickettsiae, andviruses, e.g., syphilis, chronic Trypanosoma cruzi infection); fulminantcardiac amyloidosis; heart surgery; heart transplantation; traumaticcardiac injury (e.g., penetrating or blunt cardiac injury, and aorticvalve rupture), surgical repair of a thoracic aortic aneurysm; asuprarenal aortic aneurysm; cardiogenic shock due to myocardialinfarction or cardiac failure; neurogenic shock and anaphylaxis.

“Subject” includes living organisms such as humans, monkeys, cows,sheep, horses, pigs, cattle, goats, dogs, cats, mice, rats, culturedcells therefrom, and transgenic species thereof In a preferredembodiment, the subject is a human. Administration of the compositionsof the present invention to a subject to be treated can be carried outusing known procedures, at dosages and for periods of time effective totreat the condition in the subject. An effective amount of thetherapeutic compound necessary to achieve a therapeutic effect may varyaccording to factors such as the age, sex, and weight of the subject,and the ability of the therapeutic compound to treat the foreign agentsin the subject. Dosage regimens can be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

“Substantially pure” includes compounds, e.g., drugs, proteins orpolypeptides that have been separated from components which naturallyaccompany it. Typically, a compound is substantially pure when at least10%, more preferably at least 20%, more preferably at least 50%, morepreferably at least 60%, more preferably at least 75%, more preferablyat least 90%, and most preferably at least 99% of the total material (byvolume, by wet or dry weight, or by mole percent or mole fraction) in asample is the compound of interest. Purity can be measured by anyappropriate method, e.g., in the case of polypeptides by columnchromatography, gel electrophoresis or HPLC analysis. A compound, e.g.,a protein, is also substantially purified when it is essentially free ofnaturally associated components or when it is separated from the nativecontaminants which accompany it in its natural state. Included withinthe meaning of the term “substantially pure” are compounds, such asproteins or polypeptides, which are homogeneously pure, for example,where at least 95% of the total protein (by volume, by wet or dryweight, or by mole percent or mole fraction) in a sample is the proteinor polypeptide of interest.

“Administering” includes routes of administration which allow thecompositions of the invention to perform their intended function, e.g.,treating or preventing cardiac injury caused by hypoxia or ischemia. Avariety of routes of administration are possible including, but notnecessarily limited to parenteral (e.g., intravenous, intraarterial,intramuscular, subcutaneous injection), oral (e.g., dietary), topical,nasal, rectal, or via slow releasing microcarriers depending on thedisease or condition to be treated. Oral, parenteral and intravenousadministration are preferred modes of administration. Formulation of thecompound to be administered will vary according to the route ofadministration selected (e.g., solution, emulsion, gels, aerosols,capsule). An appropriate composition comprising the compound to beadministered can be prepared in a physiologically acceptable vehicle orcarrier and optional adjuvants and preservatives. For solutions oremulsions, suitable carriers include, for example, aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media, sterile water, creams, ointments, lotions, oils,pastes and solid carriers. Parenteral vehicles can include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Intravenous vehicles can includevarious additives, preservatives, or fluid, nutrient or electrolytereplenishers (See generally, Remington's Pharmaceutical Science, 16thEdition, Mack, Ed. (1980)).

“Effective amount” includes those amounts of erythropoietin which allowit to perform its intended function, e.g., treating or preventing,partially or totally, cardiac injury caused by hypoxia or ischemia asdescribed herein. The effective amount will depend upon a number offactors, including biological activity, age, body weight, sex, generalhealth, severity of the condition to be treated, as well as appropriatepharmacolinetic properties. For example, dosages of the active substancemay be from about 0.01 mg/kg/day to about 500 mg/kg/day, advantageouslyfrom about 0.1 mg/kg/day to about 100 mg/kg/day. A therapeuticallyeffective amount of the active substance can be administered by anappropriate route in a single dose or multiple doses. Further, thedosages of the active substance can be proportionally increased ordecreased as indicated by the exigencies of the therapeutic orprophylactic situation.

“Specific binding” or “specifically binds” includes proteins, such as anantibody which recognizes and binds an erythropoietin or a ligandthereof, but does not substantially recognize or bind other molecules ina sample.

“Pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like which arecompatible with the activity of the compound and are physiologicallyacceptable to the subject. An example of a pharmaceutically acceptablecarrier is buffered normal saline (0.15M NaCl). The use of such mediaand agents for pharmaceutically active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the therapeutic compound, use thereof in the compositions suitablefor pharmaceutical administration is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

“Pharmaceutically acceptable esters” includes relatively non-toxic,esterified products of therapeutic compounds of the invention. Theseesters can be prepared in situ during the final isolation andpurification of the therapeutic compounds or by separately reacting thepurified therapeutic compound in its free acid form or hydroxyl with asuitable esterifying agent; either of which are methods known to thoseskilled in the art. Acids can be converted into esters according tomethods well known to one of ordinary skill in the art, e.g., viatreatment with an alcohol in the presence of a catalyst.

“Additional ingredients” include, but are not limited to, one or more ofthe following: excipients; surface active agents; dispersing agents;inert diluents; granulating and disintegrating agents; binding agents;lubricating agents; sweetening agents; flavoring agents; coloringagents; preservatives; physiologically degradable compositions such asgelatin; aqueous vehicles and solvents; oily vehicles and solvents;suspending agents; dispersing or wetting agents; emulsifying agents,demulcents; buffers; salts; thickening agents; fillers; emulsifyingagents; antioxidants; antibiotics; antifungal agents; stabilizingagents; and pharmaceutically acceptable polymeric or hydrophobicmaterials. Other “additional ingredients” which may be included in thepharmaceutical compositions of the invention are known in the art anddescribed, e.g., in Remington 's Pharmaceutical Sciences.

“Unit dose” includes a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient.

“Protective mechanisms” include biological events and pathways thatinhibit or reduce cellular damage caused by oxidative and/or nitrosativestress.

The invention relates in part to methods of treating or preventingmyocardial oxidative stress, such as is caused by hypoxia or ischemia,in a subject. This is done by administering to a subject in need thereoferythropoietin which modulates myocardial oxidative stress such that themyocardial cells which are the target of the oxidative stress areprotected from cell death. The cell death may be due, e.g., to necrosisor apoptosis.

The invention relates in part to methods of treating or preventingmyocardial nitrosative stress, such as is caused by drugs, infection,inflammation, hypoxia, ischemia or other causes, in a subject. This isdone by administering to a subject in need thereof erythropoietin whichmodulates myocardial nitrosative stress such that the myocardial cellswhich are the target of the nitrosative stress are protected from celldeath. The cell death may be due, e.g., to necrosis or apoptosis.

The method by which erythropoietin treatment results in cardioprotectionis thought to be independent from erythropoietin's effects on red bloodcell numbers (See Example 1; FIGS. 1-3). Therefore, the erythropoietincan be given at such a concentration that the subject's hematocrit levelis not substantially increased. A substantial increase in hematocritwould be an increase of about 10% or greater. Methods for measuring thehematocrit in a subject are known in the art, and includecentrifugation, automated cell counting, and spectroscopy.Alternatively, the erythropoietin can be given at such a concentrationthat red blood cell production is not substantially increased insubject. The present invention also encompasses methods in whicherythropoietin is given at a concentration that increasescardioprotective mechanisms without substantially increasing a subject'shematocrit level. Non-limiting examples of detectable cardioprotectivemechanisms include the Akt kinase pathway (including, e.g., signaling toendothelial nitric oxide synthase) and the ERK kinase (e.g., MEK1)pathway. Alternatively, the erythropoietin is given at the lowestconcentration wherein the erythropoietin polypeptide binds to theerythropoietin receptor (EPO-R). Methods for measuring the binding oferythropoietin to EPO-R in vivo or ex vivo are known in the art,including laser-scanning imaging, radio-ligand binding studies, flowcytometry, and agonist/antagonist studies.

Further, erythropoietin can be given for a time period in which thehematocrit of the subject is not increased. Generally, this time periodwill be about four to five days, but may be longer or shorter as neededfor treatment, and erythropoietin may be administered one or more timesper day throughout the duration of treatment. Therefore, the methods ofthis invention are useful as therapeutic and/or protective treatmentsfor subjects (e.g., humans) suffering from or at risk of cardiac injury,for whom addition of erythropoietin such that the hematocrit of thepatients is increased elevates the risk of adverse cardiac events. (SeeBesarab et al. (1998) N. Eng. J. Med. 339: 584). Therefore, the methodsof this invention are particularly useful in clinical situations inwhich it is desirable to treat or prevent a cardiac injury, but thatelevation of a patient's hematocrit levels will increase the risk ofmortality and/or morbidity.

In subjects suffering from myocardial infarction, erythropoietintreatment results in an increase in the density of the beta adrenergicreceptor (also known as beta receptor) on the surface of the leftventricle. A decrease in the concentration or activity of betareceptors, such as is known to occur during cardiac injury, requires acorresponding increase in the dosage compounds used as beta receptoragonists (e.g., dopamine, dobutamine, isoprel, and norepinephrine). Thisdosage increase in turn leads to a loss of tolerance due toover-stimulation, which decreases the effectivity of the administeredcompounds. Since erythropoietin treatment results in an increase in thedensity of the beta adrenergic receptors (See Example 2; FIG. 4), it isuseful to prevent loss of tolerance and to increase effectivity ofadministered beta receptor agonists.

Erythropoietin increases the density of beta adrenergic receptors viaone or more cell signaling pathways. Increasing the density and/oractivity of beta adrenergic receptors by administration oferythropoietin is useful to prevent or treat cardiac injury or defectsin heart rhythm (e.g., tachycardia, bradycardia from any cause, andcarotid hypersensitivity, such as due to an autonomic dysfunction).Erythropoietin administration is also useful to increase the sensitivityof cardiac tissue to anti-arrhythmic compounds and cardiac contractilityenhancing compounds.

Administration of the compounds of the invention may be done whereclinically necessary or desirable, e.g., prior to ischemia, at the onsetof ischemia, or at one or more times following the onset of ischemia.

The erythropoietin can be obtained by natural sources (e.g. urinaryerythropoietin; See U.S. Pat. No. 3,865,801). The purification of humanurinary erythropoietin by Miyake et al. in J. Biol. Chem., 252, 5558(1977), used, as starting material, urine from aplastic anemicindividuals. Naturally-occurring human erythropoietin is firsttranslated to a 166 amino acid-containing polypeptide chain witharginine 166. In a postranslational modification arginine 166 is cleavedby a carboxypeptidase. The molecular weight of the polypeptide chain ofhuman erythropoietin without the sugar moieties is 18,236 Da. In theintact erythropoietin molecule, approximately 40% of the molecularweight is accounted for by the carbohydrate groups (Sasaki, H, Bothner,B, Dell, A and Fukuda, M (1987) J. Biol. Chem. 262: 12059).

The identification, cloning, and expression of genes encodingerythropoietin are described in Egrie et al. (1986) Immunobiol. 72:213-224 and U.S. Pat. No. 4,703,008. A description of the purificationof recombinant erythropoietin from cell medium that supported the growthof mammalian cells containing recombinant erythropoietin plasmids forexample, is included in U.S. Pat. No. 4,667,016.

Instead of the recombinant erythropoietin protein, modifications of saidprotein having a higher or lower molecular weight than 34,000 Da(molecular weight of urinary erythropoietin), isoforms of the enzyme orproteins with different glycosylation may also be used. The isoforms ofurinary derived human erythropoietin are different than the isoforms ofrecombinant erythropoietin. Moreover, in principle, those proteinsderived from the amino acid sequence of natural erythropoietin with alength of 166 amino acids by way of deletions, substitutions orextensions are also possible. Essentially, such proteins havephysiological properties comparable to recombinant erythropoietin. Inparticular, such proteins have biological properties inducing the bonemarrow to increase the production of reticulocytes and red blood cellsand/or to increase hemoglobin synthesis or iron absorption. Instead ofthese proteins, low molecular weight substances may also be used, whichare referred to as erythropoietin mimetics and bind to the samebiological receptor. Preferably, these miimetics may also beadministered by the oral route. The amount of such proteins or mimeticsto be administered is determined by comparing the biological activitiesof erythropoietin and said active substances.

Erythropoietin-like polypeptides are also encompassed by the presentinvention, including, e.g., darbepoietin (from Amgen; also known asAranesp and novel erthyropoiesis stimulating protein (NESP)).Administration of darbepoietin for use in the present invention includessubcutaneous or intravenous administration at about 0.5 micrograms/kgonce a week.

The invention encompasses the preparation and use of pharmaceuticalcompositions comprising a compound, such as erythropoietin, useful forthe prevention or reduction of hypoxic/ischemic cardiac injury as anactive ingredient. Such a pharmaceutical composition may consist of theactive ingredient alone, in a form suitable for administration to asubject, or the pharmaceutical composition may comprise the activeingredient and one or more pharmaceutically acceptable carriers, one ormore additional ingredients, or some combination of these. The activeingredient may be present in the pharmaceutical composition in the formof a pharmaceutically acceptable ester or salt, such as in combinationwith a physiologically-acceptable cation or anion, as is well known inthe art. Further, the erythropoietin may contain pharmacologicallyacceptable additives (e.g., carrier, excipient and diluent), stabilizersor components necessary for formulating preparations, which aregenerally used for pharmaceutical products, as long as it does notadversely affect the efficacy of the preparation, e.g., in decreasing orinhibiting ischemia or reperfusion injury.

Examples of anti-arrhythmic compounds include, e.g., adenosine,amiodarone, bretylium, disopyramide, flecainide, lignocaine, mexiletineand propafenone.

Examples of contractility enhancing compounds include cardiac glycosidessuch as digoxin and digitoxin, sympathomimetic amines such as dobutamineand dopamine, phosphodiesterase inhibitors such as amrinone andmilrinone, compounds that increase sarcoplasmic reticulum Ca²⁺-ATPaseactivity, contractilin, isoproterenol and other compounds.

Examples of additives and stabilizers include saccharides such asmonosaccharides (e.g., glucose and fructose), disaccharides (e.g.,sucrose, lactose and maltose) and sugar alcohols (e.g., mannitol andsorbitol); organic acids such as citric acid, maleic acid and tartaricacid and salts thereof (e.g., sodium salt, potassium salt and calciumsalt); amino acids such as glycine, aspartic acid and glutamic acid andsalts thereof (e.g., sodium, calcium or potassium salt); surfactantssuch as polyethylene glycol, polyoxyethylene-polyoxypropylene copolymerand polyoxyethylenesorbitan fatty acid ester; heparin; and albumin.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions that aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates.

Pharmaceutical compositions that are useful in the methods of theinvention maybe prepared, packaged, or sold in formulations suitable fororal, rectal, vaginal, parenteral, topical, pulmonary (See e.g., U.S.Pat. No. 5,354,934), intranasal, buccal, ophthalmic, or another route ofadministration. The preferred mode is intravenous administration.

The erythropoietin and the above-mentioned ingredients are admixed asappropriate to give powder, granule, tablet, capsule, syrup, injectionand the like. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. The amount of the active ingredient is generally equal to thedosage of the active ingredient, which would be administered to asubject, or a convenient fraction of such a dosage such as, for example,one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents.

Particularly contemplated additional agents include anti-emetics andscavengers such as cyanide and cyanate scavengers. Controlled- orsustained-release formulations of a pharmaceutical composition of theinvention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include potato starch and sodium starchglycollate. Known surface active agents include sodium lauryl sulfate.Known diluents include calcium carbonate, sodium carbonate, lactose,microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include corn starch and alginic acid. Known binding agentsinclude gelatin, acacia, pre-gelatinized maize starch,polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Knownlubricating agents include magnesium stearate, stearic acid, silica, andtalc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in,e.g., U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include naturally-occurring phosphatides such aslecithin, condensation products of an alkylene oxide with a fatty acid,with a long chain aliphatic alcohol, with a partial ester derived from afatty acid and a hexitol, or with a partial ester derived from a fattyacid and a hexitol anhydride (e.g., polyoxyethylene stearate,heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, andpolyoxyethylene sorbitan monooleate, respectively). Known emulsifyingagents include lecithin and acacia. Known preservatives include methyl,ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, and sorbicacid. Known sweetening agents include glycerol, propylene glycol,sorbitol, sucrose, and saccharin. Known thickening agents for oilysuspensions include, for example, beeswax, hard paraffin, and cetylalcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e., about 20° C.) and which isliquid at the rectal temperature of the subject (i.e., about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for vaginal administration. Such acomposition may be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, a gel or cream or solution for vaginal irrigation.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e., such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made bycombining the active ingredient with a pharmaceutically acceptableliquid carrier. As is well known in the art, douche preparations may beadministered using, and may be packaged within, a delivery deviceadapted to the vaginal anatomy of the subject.

Douche preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants, antibiotics, antifungalagents, and preservatives.

Additional delivery methods for administration of compounds include adrug delivery device, such as that described in U.S. Pat. No. 5,928,195.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle (e.g., sterile pyrogen-free water) prior toparenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or diglycerides. Other parentally-administrable formulations thatare useful include those, which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and preferably from about 1 toabout 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may fer comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers. Such a formulation is administered inthe manner in which snuff is taken i.e., by rapid inhalation through thenasal passage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may frrther comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient. Suchpowdered, aerosolized, or aerosolized formulations, when dispersed,preferably have an average particle or droplet size in the range fromabout 0.1 to about 200 nanometers, and may further comprise one or moreof the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other ophthalmalmically-administrableformulations that are useful include those, which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

The mixture of erythropoietin and pharmacologically acceptable additivesis preferably prepared as a lyophilized product, and dissolved when inuse. Such preparation can be prepared into a solution containing about0.01-100.0 mg/ml of erythropoietin, by dissolving same in distilledwater for injection or sterile purified water. More preferably, it isadjusted to have a physiologically isotonic salt concentration and aphysiologically desirable pH value (pH 6-8).

Erythropoietin serum concentration is normally within the range of 5-50mU/ml. For patients suffering from chronic renal failure or otherconditions involving anemia, erythropoietin is generally administeredeither subcutaneously or intravenously at a concentration of 50-100 U/kg(a dose of 3,000-7,000 U) three times per week, or once one week priorto surgery. While the dose is appropriately determined depending onsymptom, body weight, sex, animal species and the like, it is generallyassumed that treatment options holding the blood concentration at about1-100 mU/ml will be preferred. This plasma concentration may be achievedthrough administration of one to several doses a day. Whenerythropoietin is to be administered to a subject, 0.1 ng to 10 mg/kgbody weight (e.g. 1 ng to 1 mg/kg body weight) of erythropoietin can begiven intravenously.

The compound may be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even leesfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the animal, etc.

EXAMPLES

These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Example 1 In Vivo Studies of Erythropoietin Preservation of CardiacContractility

A coronary artery ligation model was used to demonstrate the protectiveeffect of erythropoietin in the absence of an increase in hematocrit.Animals used in this study were adult male New Zealand White rabbits(3-5 kg, generally 4 kg). Animals were housed under standard conditionsand allowed to feed ad lib. The Animal Care and Use Committee of DukeUniversity approved all procedures performed in accordance with theregulations adopted by the National Institutes of Health. A myocardialinfarction (MI) was produced via the ligation of a marginal branch ofthe left circumflex coronary artery (LCx) using 5-0 prolene suture (Seee.g., Maurice et al. Am J Physiol. 276:H1853-H1860, 1999; Shah et al.,Circulation 103:1311-1316, 2001). Rabbits were anesthetized with amixture of ketamine (30 mg/kg) and acepromazine (0.5 mg/kg), intubated,and mechanically ventilated. A left thoracotomy was performed throughthe 3^(rd) or 4^(th) intercostal space, and the large marginal branch ofthe LCx was identified and ligated with a 5-0 Prolene suture. A controlgroup included sham operated animals in which only a thoracotomy andpericardiotomy were performed. Anatomic closure was performed, the chestwas evacuated of residual air using a 14-gauge angiocatheter attached toa syringe, and the animal was extubated when able to breathespontaneously. Animals were allowed to recover and returned to theircages when awake and responsive. For characterization of infarctionsize, hearts were dissected after euthanasia and the aortic root wascannulated. Each heart was rinsed with 40 cc of normal saline followedby 40 cc of triphenyltetrazolium chloride (SIGMA 8) at 37° C. The heartwas then disected at the right ventricular free wall and both atria. Theheart was sectioned in 2-3 mm segments from apex to atrio-ventriculargroove in a transverse fashion. Each segment was weighed, recorded andplaced in formalin. After six hours the specimen was digitallyphotographed in a camera mount to normalize specimen-to-lens distance.Each photograph was then appended to Adobe Photoshop (Adobe A) tomeasure pixel density of infarcted versus non infarcted areas. Thepercentage of infarction of each slide was multiplied by the mass ofeach specimen. The sum of all specimen percentages resulted in anoverall percentage of infarction in each animal.

To measure in vivo hemodynamic data in conscious animals, rabbits werelightly sedated with ketamine (30 mg/kg) and acepromazine (0.05 mg/kg).The right carotid artery was then exposed and a 2.5 Fr micromanometer(Millar Instruments) was advanced into the LV cavity to recordhemodynamics. FIG. 1 demonstrates an increase in cardiac contractilitywhen erythropoietin is administered 5 minutes after myocardial ischemiainduced by suture ligation of the large branch of the left circumflexartery. FIG. 2 demonstrates an increase in cardiac contractility whenerythropoietin is administered 24 hous prior to myocardial ischemiainduced by suture ligation of the large branch of the left circumflexartery. FIG. 3 demonstrates that hematocrit levels are not acutelyincreased four days after administration of 5,000 U/kg oferythropoietin. Similarly, administration of 1,000 U/kg oferythropoietin is cardioprotective as measured by increased cardiaccontractility, but does not acutely increase hematocrit levels four daysafter administration. Administration of lower concentrations oferythropoietin (e.g., 750 U/kg, 500 U/kg, 250 U/kg, 100 U/kg, 50 U/kg,or lower levels) are also encompassed by the present invention. Serialhematocrits were obtained on the day of erythropoietin administrationand then serially for a period of four days.

Example 2 In Vivo Studies of Erythropoietin Preservation of LeftVentricular Beta-receptor Density Following Myocardial Infarction

A beta agonist receptor ligand binding assay was used to demonstrate themaintenance of left ventricular beta receptor levels followingadministration of erythropoietin. Myocardial membranes were preparedfrom frozen hearts (See, e.g., Maurice et al. Am J Physiol.276:H1853-H1860, 1999). Final purified cardiac membranes were suspendedat a concentration of 1-2 mg/ml and receptor binding was performed usingthe nonselective βAR ligand [¹²⁵I] cyanopindolol. Nonspecific bindingwas determined in the presence of 20 μM alprenolol. All assays wereperformed in triplicate, and receptor density (measured in fmoles) wasnormalized to mg of membrane protein. As shown in FIG. 4, myocardialinfarction causes a reduction in cardiac beta receptor density, which ismitigated by treatment with erytlropoietin. Administration of anyconcentration of erythropoietin that does not increase hematocrit levelsis useful to inhibit or decrease the reduction in cardiac beta receptordensity caused by cardiac events, such as myocardial infarction.Specifically, erythropoietin can be administered at concentrations of5,000 U/kg, 1,000 U/kg, 750 U/kg, 500 U/kg, 250 U/kg, 100 U/kg, 50 U/kg,or lower levels).

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. Various substitutions, alterations, and modificationsmay be made to the invention without departing from the spirit and scopeof the invention as defined by the claims. Other aspects, advantages,and modifications are within the scope of the invention. The contents ofall references, issued patents, and published patent applications citedthroughout this application are hereby incorporated by reference.

1. A method of treating or preventing myocardial oxidative stress in asubject, comprising administering erythropoietin to a subject in needthereof at a concentration that does not increase the hematocrit in saidsubject.
 2. The method of claim 1, wherein said myocardial oxidativestress is caused by hypoxia or ischemia.
 3. The method of claim 1,wherein said erythropoietin is administered at a concentration of lessthan about 5,000 U/kg.
 4. A method of treating or preventing myocardialoxidative stress in a subject, comprising administering erythropoietinto a subject in need thereof at a concentration that does not induce redblood cell production in said subject.
 5. The method of claim 4, whereinsaid erythropoietin is administered at a concentration of less thanabout 5,000 U/kg.
 6. A method of treating or preventing myocardialoxidative stress in a subject, comprising administering erythropoietinto a subject in need thereof for a time period in which the hematocritin said subject is not increased.
 7. The method of claim 6, wherein saidtime period is about one week.
 8. The method of claim 6, wherein saidtime period is about four days.
 9. A method of treating or preventingmyocardial nitrosative stress in a subject, comprising administeringerythropoietin to a subject in need thereof at a concentration that doesnot increase the hematocrit in said subject.
 10. The method of claim 9,wherein said myocardial nitrosative stress is caused by drugs,infection, inflammation, hypoxia or ischemia.
 11. The method of claim 9,wherein said erythropoietin is administered at a concentration of lessthan about 5,000 U/kg.
 12. A method of treating or preventing myocardialnitrosative stress in a subject, comprising administering erythropoietinto a subject in need thereof at a concentration that does not induce redblood cell production in said subject.
 13. The method of claim 12,wherein said erythropoietin is administered at a concentration of lessthan about 5,000 U/kg.
 14. A method of treating or preventing myocardialnitrosative stress in a subject, comprising administering erythropoietinto a subject in need thereof for a time period in which the hematocritin said subject is not increased.
 15. The method of claim 14, whereinsaid time period is about one week.
 16. The method of claim 14, whereinsaid time period is about four days.
 17. A method of modulating acardioprotective signaling pathway, comprising administering, to asubject in need of cardioprotection, erythropoietin at a concentrationthat does not induce red blood cell production in said subject.
 18. Themethod of claim 17, wherein said cardioprotective signaling pathway isselected from the group consisting of MAP kinase, PI3 kinase, aninsulin-responsive pathway, hormones, ischemia preconditioning,adenosine pathways, ras, JAK/STAT, nitric oxide synthase, hemoxygenase,xanthine oxidase, NADPH oxidase, cytochrome p450, cytochrome p450reductase, oxigenases, denitrosylases, GSNO reductase, oxygen-carryingproteins, nitric oxide-carrying proteins, and carbon monoxide-carryingproteins.
 19. A method of treating or preventing cardiac injury in asubject, comprising administering erythropoietin to a subject in needthereof at a concentration that does not increase the hematocrit in saidsubject.
 20. The method of claim 19, wherein said cardiac injury iscaused by hypoxia or ischemia.
 21. The method of claim 19, wherein saidinjury is selected from the group consisting of myocardial infarction,cardiac arrest, ischemia-reperfusion injury, congestive heart failure,cardiotoxicity, cardiac damage due to parasitic infection, fulminantcardiac amyloidosis, heart surgery, heart transplantation, traumaticcardiac injury, surgical repair of a thoracic aortic aneurysm, asuprarenal aortic aneurysm, hemorrhagic shock due to blood loss,cardiogenic shock due to myocardial infarction or cardiac failure, andanaphylaxis.
 22. The method of claim 19, wherein said administration isdone prior to reperfusion or infarction.
 23. The method of claim 19,wherein said administration is done at the onset of reperfusion.
 24. Themethod of claim, 19, wherein said administration is done subsequent toinfarction or cardiac damage.
 25. The method of claim 19, wherein thesubject also suffers from end-stage renal disease or diabetes.
 26. Amethod of treating or preventing cardiac injury in a subject, comprisingadministering erythropoietin to a subject in need thereof at aconcentration that does not induce red blood cell production in saidsubject.
 27. The method of claim 26, wherein said cardiac injury iscaused by hypoxia or ischemia.
 28. The method of claim 26, wherein saidinjury is selected from the group consisting of myocardial infarction,cardiac arrest, ischemia-reperfusion injury, congestive heart failure,cardiotoxicity, cardiac damage due to parasitic infection, fulminantcardiac amyloidosis, heart surgery, heart transplantation, traumaticcardiac injury, surgical repair of a thoracic aortic aneurysm, asuprarenal aortic aneurysm, hemorrhagic shock due to blood loss,cardiogenic shock due to myocardial infarction or cardiac failure,anaphylaxis, unstable coronary syndrome, tachycardia and bradycardia.29. The method of claim 26, wherein said administration is done prior toreperfusion.
 30. The method of claim 26, wherein said administration isdone at the onset of reperfusion.
 31. The method of claim 26, whereinthe subject also suffers from end-stage renal disease or diabetes.
 32. Amethod of treating or preventing cardiac injury in a subject, comprisingadministering erythropoietin to a subject in need thereof for a timeperiod in which the hematocrit in said subject is not increased.
 33. Themethod of claim 32, wherein said time period is about one week.
 34. Themethod of claim 32, wherein said time period is about four days.
 35. Themethod of claim 32, wherein said injury is selected from the groupconsisting of myocardial infarction, cardiac arrest,ischemia-reperfusion injury, congestive heart failure, cardiotoxicity,cardiac damage due to parasitic infection, fulminant cardiacamyloidosis, heart surgery, heart transplantation, traumatic cardiacinjury, surgical repair of a thoracic aortic aneurysm, a suprarenalaortic aneurysm, hemorrhagic shock due to blood loss, cardiogenic shockdue to myocardial infarction or cardiac failure, anaphylaxis, unstablecoronary syndrome, tachycardia and bradycardia
 36. A method ofpreventing organ or tissue damage during organ or tissuetransplantation, comprising administering to an organ donorerythropoietin at a concentration that does not increase the hematocritin said donor, prior to or concurrent with removal of said organ. 37.The method of claim 36, wherein said organ is the heart.
 38. A method oftreating heart failure in a subject, comprising treating said subjectwith erythropoietin at a concentration that does not increase thehematocrit in said subject and a compound selected from the groupconsisting of anti-platelet drugs, anti-coagulant drugs, anti-thromboticdrugs, anti-oxidants, anti-nitrosants, cholesterol lowering drugs,aspirin and aspirin-related derivatives.
 39. A method of treating asurvivor of a myocardial infarction, comprising administeringerythropoietin at a concentration that does not increase the hematocritin said survivor, wherein the erythropoietin is administered in a singledose within 3 hours of the myocardial infarction.
 40. The method ofclaim 39, when the erythropoietin is administered in a single dosewithin 1 hour of the myoc dial infarction.
 41. The method of claim 39,wherein the erythropoietin is administered in a single dose within 5minutes of the myocardial infarction.
 42. The method of claim 39,further comprising administering to said survivor a compound selectedfrom the group consisting of an angiotensin-converting enzyme inhibitor,GLP-1, beta blockers, thrombolytics, ADP receptor antagonists,anti-platelet drugs, anti-coagulant drugs, anti-thrombotic drugs,anti-oxidants, anti-nitrosants, cholesterol lowering drugs, aspirin andaspirin-related derivatives.
 43. The method of claim 39, wherein thesurvivor also suffers from end-stage renal failure or diabetes.
 44. Amethod of treating a survivor of a myocardial infarction, comprisingadministering erythropoietin at a concentration that does not increasethe hematocrit in said survivor, wherein the erythropoietin isadministered for an extended period of time.
 45. The method of claim 44,wherein said administration is continuous.
 46. The method of claim 44,further comprising administering to said survivor a compound selectedfrom the group consisting of an angiotensin-converting enzyme inhibitor,GLP-1, beta blockers, thrombolytics, ADP receptor antagonists,anti-platelet drugs, anti-coagulant drugs, anti-thrombotic drugs,anti-oxidants, anti-nitrosants, cholesterol lowering drugs, aspirin andaspirin-related derivatives.
 47. The method of claim 44, wherein thesurvivor also suffers from end-stage renal failure or diabetes.
 48. Amethod of preventing or reducing the severity of ischemia-reperfusioninjury in a subject at risk for ischemia-reperfusion injury comprisingadministering to the subject an amount of erythropoietin at aconcentration that does not increase the hematocrit of the subject. 49.The method of claim 48, wherein the subject is at risk forischemia-reperfusion injury due to undergoing treatment for myocardialinfarction, cardiac arrest, ischemia-reperfusion injury, congestiveheart failure, cardiotoxicity, cardiac damage due to parasiticinfection, fulminant cardiac amyloidosis, heart surgery, hearttransplantation, traumatic cardiac injury, surgical repair of a thoracicaortic aneurysm, a suprarenal aortic aneurysm, hemorrhagic shock due toblood loss, cardiogenic shock due to myocardial infarction or cardiacfailure, anaphylaxis, a suprarenal aortic aneurysm, liver, kidney, smallintestine, or pancreas transplant, hepatic and biliary surgicalresections, total or partial pancreatectomy, total and partialgastrectomy, esophagectomy, colorectal surgery, vascular surgery formesenteric vascular disease, abdominal insufflation during laparoscopicsurgical procedures, blunt or penetrating trauma to the abdomenincluding gun shot wounds, stab wounds or penetrating wounds or bluntabdominal trauma secondary to deceleration injury or motor vehicleaccidents and neurogenic shock.
 50. The method of claim 48, whereinerythropoietin is administered to the subject prior to undergoingtreatment.
 51. The method of claim 48, wherein erythropoietin isadministered to the subject subsequent to undergoing treatment.
 52. Amethod of pre-conditioning a subject at risk for a cardiac injury due toa surgical procedure, comprising administering to the subject an amountof erythropoietin at a concentration that does not increase thehematocrit of the subject prior to the surgical procedure.
 53. Themethod of claim 52, wherein erythropoietin is administered to thesubject at least about once per day for about seven days prior to saidsurgical procedure.
 54. The method of claim 52, wherein erythropoietinis administered to the subject at least about one day prior to saidsurgical procedure.
 55. A method of pre-conditioning a subject at riskfor a cardiac injury due to a surgical procedure, comprisingadministering to the subject an amount of erythropoietin for a timeperiod in which the hematocrit in said subject is not increased.
 56. Themethod of claim 55, wherein said time period is about one week.
 57. Themethod of claim 55, wherein said time period is about four days.
 58. Themethod of claim 55, wherein erythropoietin is administered to thesubject at least about once per day for about three days prior to saidsurgical procedure.
 59. The method of claim 55, wherein erythropoietinis administered to the subject at least about one day prior to saidsurgical procedure.
 60. A method of increasing beta-receptor density ina subject suffering from cardiac injury, comprising administeringerythropoietin at a concentration that does not increase the hematocritin said subject.
 61. The method of claim 60, wherein said cardiac injuryis selected from the group consisting of myocardial infarction andcardiac stunning from ischemia or apoptosis.
 62. The method of claim 60,wherein the erythropoietin is administered for an extended period oftime.
 63. A method of preserving beta-receptor sensitivity in a subjectsuffering from cardiac injury, comprising administering erythropoietinat a concentration that does not increase the hematocrit in saidsubject.
 64. A method of preserving beta-receptor sensitivity in asubject suffering from cardiac injury, comprising administeringerythropoietin at a concentration that does not induce red blood cellproduction.
 65. A method of preventing reduced sensitivity to one ormore cardiostimulatory compounds in a subject suffering from or at riskof a cardiac injury, comprising administering erythropoietin at aconcentration that does not increase the hematocrit in said subject. 66.The method of claim 65, wherein said cardiostimulatory compounds areselected from the group consisting of anti-arrhythmic compounds andcontractility enhancing compounds.
 67. The method of claim 65, whereinsaid cardiostimulatory compounds are selected from the group consistingof dopamine, dobutamine, isoprel, digoxin, digitoxin, andnorepinephrine.
 68. A method of increasing sensitivity to one or morecardiostimulatory compounds in a subject suffering from or at risk of acardiac injury, comprising administering erythropoietin at aconcentration that does not increase the hematocrit in said subject. 69.The method of claim 68, wherein said cardiostimulatory compounds areselected from the group consisting of anti-arrhythmic compounds andcontractility enhancing compounds.
 70. The method of claim 68, whereinsaid cardiostimulatory compounds are selected from the group consistingof dopamine, dobutamine, isoprel, digoxin, digitoxin, andnorepinephrine.
 71. A method of increasing beta-receptor density in asubject suffering from cardiac injury, comprising administering to thesubject an amount of erythropoietin for a time period in which thehematocrit in said subject is not increased.
 72. The method of claim 71,wherein said time period is about one week.
 73. The method of claim 71,wherein said time period is about four days.